PROJECT SUMMARY EpiCypher is pioneering the commercial development of fully defined recombinant / semi-synthetic nucleosomes carrying specific histone post-translational modifications (PTMs) (termed designer nucleosomes or dNucsTM) for epigenetics research. We currently use native chemical ligation (NCL) to generate modified histones for dNuc assembly, and have developed proprietary methods to efficiently manufacture 2-10 mg histone lots carrying single or combinatorial PTMs on H2B, H2A, H3 or H4; we are currently applying these to dNuc- based applications, including several high-throughput inhibitor screening platforms (AlphaNucTM and EpiDyneTM), and the first qualitative / quantitative ChIP platform (SNAP-ChIPTM). Despite our optimization of dNuc manufacturing, the approaches are multistep, labor-intensive, and requires four weeks of dedicated effort to synthesize each modified-histone in the milligram scale (plus three more weeks for octamer / nucleosome assembly and associated QC). As such, this pipeline is unsuitable for the manufacturing of combinatorial diversity nucleosome panels for discovery-based drug development and basic research applications. Here, EpiCypher is developing a protein engineering tool for accelerated dNuc manufacturing. Our innovative approach uses the Sortase A (SrtA) transpeptidase to ligate modified peptides directly onto fully assembled tailless nucleosomes (tNucs), thus generating verSaNucsTM. While NCL is optimized to generate large quantities of highly pure histones / dNucs for applications requiring homogeneous PTM-defined substrates, the verSaNuc approach enables two newfound capabilities: a) the rapid development of modified nucleosomes in small batch (g scale vs. mg for NCL); and, b) the ability to multiplex modified nucleosome syntheses. These newfound capabilities are significant as NCL lacks the throughput to deliver nucleosome substrates covering the vast PTM diversity (either of combinatorial or lesser studied marks) observed in vivo. Thus, verSaNuc manufacturing is directly complementary to our NCL approach. In Phase I, we have developed a ligation strategy to rapidly generate verSaNucs with modifications on histone H3. We showed that verSaNucs are generally functionally equivalent in many downstream assays when compared with dNucs made using NCL. In Phase II, we will use our verSaNuc manufacturing strategy to rapidly generate a diverse collection of high quality singly- and combinatorially-modified nucleosomes (Aim 1), and functionally validate these biochemical substrates using two proprietary nucleosome-based assay platforms (AlphaNucTM and EpiDyneTM; Aim 2). Also, we will leverage the ability to multiplex verSaNuc manufacturing to develop verSaScreenTM, a high-throughput discovery platform containing verSaNucs in 96-well format to rapidly decipher the combinatorial histone code (Aim 3). This technology will enable the development of next-generation nucleosome-based tools and the expansion of modified nucleosomes into new exploratory / discovery markets.